Process for the preparation of d-pantothenic acid and/or salts thereof

ABSTRACT

The invention provides a process for the preparation of D-pantothenic acid and/or salts thereof or feedstuffs additives comprising these by fermentation of microorganisms of the  Bacillus  group, in particular those which already produce D-pantothenic acid, which comprises enhancing, in particular over-expressing, in the microorganisms one or more of the nucleotide sequence(s) which code(s) for the gene or ORF ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI.

FIELD OF THE INVENTION

[0001] This invention relates to a process for the preparation ofD-pantothenic acid and/or salts thereof or mixtures comprising theseusing microorganisms of the Bacillus group in which at least one or moreof the genes or open reading frames (ORF) chosen from the groupconsisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS,adk, yusH, yqhJ, yqhK And yqhI is or are enhanced.

PRIOR ART

[0002] Pantothenic acid is produced worldwide in an order of magnitudeof several thousand ton a year. It is used inter alia in human medicine,in the pharmaceuticals industry and in the foodstuffs industry. A largeportion of the pantothenic acid produced is used for nutrition of stockanimals such as poultry and pigs.

[0003] Pantothenic acid can be prepared by chemical synthesis, orbiotechnologically by fermentation of suitable microorganisms insuitable nutrient solutions. In the chemical synthesis, DL-pantolactoneis an important precursor. It is prepared in a multi-stage process fromformaldehyde, isobutylaldehyde and cyanide, and in further processsteps, the racemic mixture is separated, D-pantolactone is subjected toa condensation reaction with β-alanine, and D-pantothenic acid isobtained in this way.

[0004] A typical commercial form is the calcium salt of D-pantothenicacid. The calcium salt of the racemic mixture of D,L-pantothenic acid isalso customary.

[0005] The advantage of the fermentative preparation by microorganismslies in the direct formation of the desired stereoisomeric form, that isto say the D-form, which is free from L-pantothenic acid.

[0006] Various species of bacteria, such as e.g. Escherichia coli (E.coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes,Brevibacterium ammoniagenes, Corynebacterium glutamicum, Bacillussubtilis-and also yeasts, such as e.g. Debaromyces castellii can produceD-pantothenic acid.

[0007] Instructions for improving the fermentative production processesare, for example, EP-A 0 493 060, EP-A-0590857, U.S. Pat. No. 5,518,906,WO97/10340, WO01/21772 or U.S. Pat. No. 6,184,007.

[0008] After fermentation, the D-pantothenic acid or the correspondingsalt is isolated from the fermentation broth and purified (EP-A-0590857and WO96/33283). The fermentation broth-containing D-pantothenic acidcan also be dried with the biomass produced during the fermentation(U.S. Pat. No. 6,238,714) and then used in particular as a feedstuffsadditive.

OBJECT OF THE INVENTION

[0009] The inventors had the object of providing new measures forimproved fermentative preparation of D-pantothenic acid and/or saltsthereof, and animal feedstuffs additives comprising these.

SUMMARY OF THE INVENTION

[0010] When D-pantothenic acid or pantothenic acid or pantothenate arementioned in the following text, this means not only the free acids butalso the salts of D-pantothenic acid, such as e.g. the calcium, sodium,ammonium or potassium salt.

[0011] The invention provides a process for the preparation ofD-pantothenic acid and/or salts thereof using microorganisms of theBacillus group which in particular already produce D-pantothenic acidand in which at least one or more of the nucleotide sequence(s) whichcode(s) for the ybbT-ORF, ywkA-ORF, yjmC-ORF, ytsJ-ORF, mdh gene, cysKgene, iolJ gene, pdhD gene, yuiE-ORF, dhaS gene, adk gene, yusH-ORF,yqhJ-ORF, yqhK-ORF and yqhI-ORF is or are enhanced, in particularover-expressed.

[0012] In particular, the process is a process which comprises carryingout the following steps:

[0013] a) fermentation of microorganisms of the Bacillus group in whichat least one or more of the genes or open reading frames chosen from thegroup consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE,dhaS, adk, yusH, yqhJ, yqhK: and yqhI is or are enhanced, in particularover-expressed, optionally in combination with the attenuation orenhancement of further genes or open reading frames,

[0014] b) optionally in the presence of alkaline earth metal compounds,these being added to the fermentation broth continuously ordiscontinuously in preferably stoichiometric amounts

[0015] c) concentration of the D-pantothenic acid or the correspondingsalts in the medium or the fermentation broth or optionally in the cellsof the microorganisms of the Enterobacteriaceae family and

[0016] d) after conclusion of the fermentation, isolation of theD-pantothenic acid, and/or of the corresponding salt(s).

[0017] The invention also provides a process in which, after conclusionof the fermentation, all or some (0 to 100%) of the biomass remains inthe fermentation broth, and the broth obtained in this way is processed,optionally after concentration, to a solid mixture which comprisesD-pantothenic acid and/or salts thereof and optionally comprises furtherconstituents of the fermentation broth.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, of the openreading frame (ORF) or ORFs, using a potent promoter or a gene or alleleor ORF which codes for a corresponding enzyme or protein with a highactivity, and optionally combining these measures.

[0019] Open reading frame (ORF) describes a section of a nucleotidesequence which codes or can code for a protein or polypeptide orribonucleic acid to which no function can be assigned according to theprior art. After assignment of a function to the nucleotide sequencesection in question, it is in general referred to as a gene.

[0020] By enhancement measures, in particular over-expression, theactivity or concentration of the corresponding protein is in generalincreased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%or 500%, up to a maximum of 1000% or 2000%, based on that of thewild-type protein or the activity or concentration of the protein in thestarting microorganism.

[0021] The microorganisms which the present invention provides canproduce D-pantothenic acid from glucose, sucrose, lactose, fructose,maltose, molasses, starch, cellulose or from glycerol and ethanol. Theyare representatives of the Bacillus group, in particular of the genusBacillus, preferably the species Bacillus subtilis.

[0022] The Bacillus group includes, inter alia, Bacillus subtilis,Bacillus lentimorbus, Bacillus lentus, Bacillus firmus, Bacilluspantothenticus, Bacillus amyloliquefaciens, Bacillus cereus, Bacilluscirculans, Bacillus coagulans, Bacillus licheniformis, Bacillusmegaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillushalodurans, Bacillus brevis, Bacillus stearothermophilus Bacillus, andother so-called group 1 Bacillus species which are characterized by thecorresponding 16S rRNA type (Priest (1993), In: Bacillus subtilis andOther Gram-Positive Bacteria, eds. Sonenshein et al., ASM, Washington,D.C., USA).

[0023] Suitable D-pantothenic acid-producing strains of the Bacillusgroup, in particular of the species Bacillus subtilis, are inter alia,for example, the strains mentioned in WO01/21772

[0024]Bacillus subtilis strain PA 221

[0025]Bacillus subtilis strain PA 248

[0026]Bacillus subtilis strain PA 236

[0027]Bacillus subtilis strain PA 221/pAN429-4

[0028]Bacillus subtilis strain PA 413-4

[0029]Bacillus subtilis strain PA 236-1

[0030]Bacillus subtilis strain PA 340

[0031]Bacillus subtilis strain PA 377

[0032]Bacillus subtilis strain PA 365

[0033]Bacillus subtilis strain PA 377-2

[0034]Bacillus subtilis strain PA 824-2.

[0035] It has been found that microorganisms of the Bacillus groupproduce D-pantothenic acid in an improved manner after enhancement, inparticular over-expression, of one or more of the genes or ORFs, or ofthe nucleotide sequences which code for these, chosen from the groupconsisting ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS,adk, yusH, yqhJ, yqhK and yqhI.

[0036] The nucleotide sequences of the genes or open reading frames(ORF) of Bacillus subtilis belong to the prior art and can also be foundin the genome sequence of Bacillus subtilis published by Kunst et al.(Nature 390, 249-256 (1997).

[0037] ybbT-ORF:

[0038] Description: Open reading frame of unknown function, similaritywith phosphoglucomutase

[0039] Reference: Kunst et al.; Nature 390:237-8 (1997)

[0040] Accession No.: Z99104

[0041] ywkA-ORF

[0042] Description: Open reading frame of unknown function, similaritywith malate dehydrogenase

[0043] Reference: Kunst et al., Nature 390: 249-256 (1997)

[0044] Accession No.: Z99122, Z99123

[0045] yjmC-ORF

[0046] Description: open reading frame of unknown function, similaritywith malate dehydrogenase from Methanococcus jannaschii

[0047] Reference: Kunst et al., Nature 390: 249-256 (1997) AccessionNo.: AF015825, Z99110

[0048] ytsJ-ORF

[0049] Description: Open reading frame of unknown function, similaritywith malate dehydrogenase (NADP+) from Anas platyrhynchos

[0050] Reference: Abe et al., Microbiology 141, 1433-1442 (1995)

[0051] Accession No.: AP008220

[0052] mdh gene:

[0053] Description: Malate dehydrogenase

[0054] EC No.: 1.1.1.37

[0055] Alternative gene name: citH

[0056] Reference: Jin et al.; Journal of Bacteriology 178:560-563 (1996)

[0057] Accession No.: AF008220

[0058] cysK gene:

[0059] Description: Cysteine synthase A Synonyms: Cysteine synthetase A,O-acetylserine sulfhydrylase A, O-acetylserine (thiol)-lyase, (CSase),superoxide inducible protein 11, (SOI11)

[0060] EC No.: 4.2.99.8

[0061] Reference: Ogasawara et al.; DNA Research 1:1-14 (1994)

[0062] Accession No.: Z99104

[0063] iolJ gene:

[0064] Description: Fructose 1,6-bisphosphate aldolase (class II)

[0065] Alternative gene names: fbaB, yxdI, alf2,

[0066] EC No.: 4.1.2.13

[0067] Reference: Yoshida et al.; Microbiology 140:2289-2298 (1994)

[0068] Accession No.: Z99124

[0069] pdhD gene:

[0070] Description: Dihydrolipoamide dehydrogenase E3 subunit both ofthe pyruvate dehydrogenase and of the 2-oxoglutarate dehydrogenasecomplex

[0071] Alternative gene names: citL, dld1, aced

[0072] EC No.: 1.8.1.4

[0073] Reference: Hemila et al., Journal of Bacteriology 172: 5052-5063(1990)

[0074] Accession No.: AF012285

[0075] yuiE-ORF:

[0076] Description: Open reading frame of unknown function, similaritywith leucyl aminopeptidase, leucine aminopeptidase (LAP)

[0077] EC No.: 3.4.11.1

[0078] Reference: Kunst et. al., Nature 390:237-8 (1997)

[0079] Accession No.: Z99120

[0080] dhaS gene

[0081] Description: Aldehyde dehydrogenase

[0082] Reference: Kunst et al., Nature 390:237-8 (1997)

[0083] Accession No.: AF027868, Z99114

[0084] adk gene:

[0085] Description: Adenylate kinase, ATP-AMP transphosphorylase,superoxide inducible protein 16 (SOI16)

[0086] EC No.: 2.7.4.3

[0087] Reference: Nakamura et al.; Journal of Biochemistry 107:603-607(1990)

[0088] Accession No.: Z99104

[0089] yusH-ORF

[0090] Description: Open reading frame of unknown function, similaritywith protein H of the glycine-cleavage system Alternative gene name:gcvH

[0091] Reference: Kunst et al., Nature 390:237-8 (1997)

[0092] Accession No.: Z99120

[0093] yqhJ-ORF

[0094] Description: Open reading frame of unknown function, similaritywith subunit 1 of glycine dehydrogenase/decarboxylase or of protein P ofthe glycine cleavage system

[0095] Alternative gene names: gcvP, gcvPA, gcs1

[0096] EC No.: 1.4.4.2

[0097] Reference: Kunst et al., Nature 390: 249-256 (1997)

[0098] Accession No.: Z99116

[0099] yqhK-ORF

[0100] Description: Open reading frame of unknown function, similaritywith subunit 2 of glycine dehydrogenase/decarboxylase or of protein P ofthe glycine cleavage system

[0101] Alternative gene names: gcvP, gcvPB, gcs2

[0102] EC No.: 1.4.4.2

[0103] Reference: Kunst et al., Nature 390: 249-256 (1997)

[0104] Accession No.: Z99116

[0105] yqhI-ORF

[0106] Description: Open reading frame of unknown function, similaritywith the aminomethyl transferase of protein T of the glycine cleavagesystem

[0107] Alternative gene names: gcvT, gcsT

[0108] EC No.: 2.1.2.10

[0109] Reference: Kunst et. al., Nature 390: 249-256 (1997)

[0110] Accession No.: Z99116

[0111] The nucleic acid sequences can be found in the databanks of theNational Center for Biotechnology Information (NCBI) of the NationalLibrary of Medicine (Bethesda, Md., USA), the nucleotide sequencedatabank of the European Molecular Biologies Laboratories (EMBL,Heidelberg, Germany or Cambridge, UK) or the DNA databank of Japan(DDBJ, Mishima, Japan). The “SubtiList” sequence databank of the PasteurInstitute (Paris, France) can furthermore be used.

[0112] The genes or open reading frames described in the text referencesmentioned can be used according to the invention. Alleles of the genesor open reading frames which result from the degeneracy of the geneticcode or due to sense mutations of neutral function can furthermore beused.

[0113] In the same way, nucleic acids or polynucleotides which code forproteins or polypeptides which are identical, homologous or similar tothe extent of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100%, originate from strains of the Bacillus group and havethe corresponding function can be used.

[0114] To achieve an over-expression, the number of copies of thecorresponding genes can be increased, or the promoter and regulationregion or the ribosome binding site upstream of the structural gene canbe mutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same way. By inducible promoters, it isadditionally possible to increase the expression in the course offermentative D-pantothenic acid production. The expression is likewiseimproved by measures to prolong the life of the m-RNA. Furthermore, theenzyme activity is also increased by preventing the degradation of theenzyme protein. The genes or gene constructs can either be present inplasmids with a varying number of copies, or can be integrated andamplified in the chromosome. Alternatively, an over-expression of thegenes in question can furthermore be achieved by changing thecomposition of the media and the culture procedure.

[0115] A strain transformed with one or more plasmid vectors, inparticular expression vectors, wherein the plasmid vector(s) carries(carry) at least one of the nucleotide sequences which code for thegenes or open reading frames ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ,pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI, can be employed in aprocess according to the invention.

[0116] In a further process according to the invention, the ribosomebinding sites of one or more of the genes or open reading frames chosenfrom the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ,pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI can be optimized.

[0117] In a further process according to the invention, an additionalpromoter can be placed before one or more of the genes or open readingframes chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh,cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI.

[0118] In a further process according to the invention, at least onefurther copy of one or more of the genes or open reading frames chosenfrom the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ,pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI can be added to thechromosome of the host.

[0119] It may furthermore be advantageous for the production ofD-pantothenic acid with strains of the Bacillus group, in addition toenhancement of one or more of the genes or open reading frames chosenfrom the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ,pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK And yqhI, for one or more of thegenes chosen from the group consisting of

[0120] the panE gene which codes for ketopantoate reductase (WO/0121772)

[0121] the polypeptide coded by the open reading frame ylbQ or the apbAgene (Kunst et. al. Nature 20; 390(6657):249-256 (1997); Accession No.Z99111)

[0122] the panB gene which codes for ketopantoate hydroxymethyltransferase (Sorokin et al., Microbiology 142:2005-2016 (1996);WO01/21772; Accession No.: L47709)

[0123] the panD gene which codes for aspartate 1-decarboxylase (Sorokinet al., Microbiology 142:2005-2016 (1996); WO01/21772; Accession No.:L47709)

[0124] the panC gene which codes for pantothenate synthetase (Sorokin etal., Microbiology 142:2005-2016 (1996); WO01/21772; Accession No.:L47709)

[0125] the ilvB and ilvN genes which code for acetohydroxy-acidsynthetase (Wipat et. al., Microbiology 142:3067-3078 (1996); AccessionNo.: Z75208)

[0126] the alsS gene which codes for α-acetolactate synthase (Renna etal., Journal of Bacteriology 175:3863-3875 (1993); Accession No.:Z93767)

[0127] the ilvC gene which codes for acetohydroxy-acid isomeroreductase(Wipat et. al., Microbiology 142:3067-3078 (1996); Accession No.:Z75208)

[0128] the ilvD gene which codes for dihydroxy-acid dehydratase (Sorokinet al., Microbiology 142:2005-2016 (1996); Accession No.: Z99115)

[0129] the serA gene which codes for phosphoglycerate dehydrogenase(Sorokin et al., Molecular Microbiology 10:385-395 (1993); AccessionNo.: L47648)

[0130] the serC gene which codes for phosphoserine amino transferase(Noback et. al., Microbiology 144:859-875 (1998); Sorokin et al.,Molecular Microbiology 10:385-395 (1993); Accession No.: Z99109)

[0131] the open reading frame ywpJ (Presecan et al. Microbiology 143:3313-3328 (1997); Accession No.: Z83337)

[0132] the glyA gene which codes for serine hydroxymethyl transferase(Kunst et. al., Nature 390: 249-256 (1997); Accession No.: Z99122)

[0133] to be enhanced, in particular over-expressed, individually ortogether.

[0134] Finally, it may be advantageous for the production ofD-pantothenic acid with strains of the Bacillus group, in addition toenhancement of one or more of the genes or open reading frames chosenfrom the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ,pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI, ornucleotide:sequences which code for these, for one or more of the genesor open reading frames chosen from the group consisting of

[0135] the protein coded by ywaA-ORF (Glaser et al., MolecularMicrobiology 10:371-384 (1993); Tobisch et al. Journal of Bacteriology179: 496-506 (1997); Accession No. Z49992)

[0136] the protein coded by ybgE-ORF (Kunst et al., Nature 390, 249-256(1997); Accession No. Z99105)

[0137] the ansB gene which codes for L-aspartase (Sun and Seflow,Journal of Bacteriology 173:3831-3845 (1991); Accession No.: D84432)

[0138] the alsD gene which codes for acetolactate decarboxylase (Rennaet al., Journal of Bacteriology 175:3863-3875 (1993); Accession No.:Z93767)

[0139] the coaA gene which codes for pantothenic acid kinase, oryqjS-ORF (Kunst et al., Nature 390, 249-256 (1997); Accession No.:Z99116)

[0140] the coaX gene which codes for coax-pantothenic acid kinase, oryacB-ORF (Kunst et al., Nature 390, 249-256 (1997); Accession No.:Z99104; WO01/21772)

[0141] to be attenuated, in particular eliminated or expressed at a lowlevel, individually or together.

[0142] The term “attenuation” in this connection describes the reductionor elimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele or ORFwhich codes for a corresponding enzyme (protein) with a low activity orinactivates the corresponding gene or ORF or enzyme (protein) andoptionally combining these measures.

[0143] The reduction in gene expression can take place by suitableculturing, by genetic modification (mutation) of the signal structuresof gene expression or also by the antisense-RNA technique. Signalstructures of gene expression are, for example, repressor genes,activator genes, operators, promoters, attenuators, ribosome bindingsites, the start codon and terminators.

[0144] Mutations which lead to a change or reduction in the catalyticproperties or activities of proteins or enzymes are known from the priorart.

[0145] Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theactivity, “missense mutations” or “nonsense mutations” are referred to.Insertions or deletions of at least one base pair in a gene lead to“frame shift mutations”, which lead to incorrect amino acids beingincorporated or translation being interrupted prematurely. If a stopcodon is formed in the coding region as a consequence of the mutation,this also leads to a premature termination of the translation. Deletionsof several codons typically lead to a complete loss of the activity.

[0146] By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

[0147] It may furthermore be advantageous for the production ofD-pantothenic acid, in addition to enhancement of one or more of thegenes or open reading frames chosen from the group consisting of ybbT,ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ,yqhK and yqhI, to eliminate undesirable side reactions (Nakayama:“Breeding of Amino Acid Producing Microorganisms”, in: Overproduction ofMicrobial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,London, UK, 1982). Bacteria in which the metabolic pathways which reducethe formation of D-pantothenic acid are at least partly eliminated canbe employed in the process according to the invention.

[0148] The microorganisms produced according to the invention can becultured in the batch process (batch culture), the fed batch (feedprocess) or the repeated fed batch process (repetitive feed process). Asummary of known culture methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung die Bioverfahrenstechnik (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

[0149] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). The media described inWO01/21772 can also be used. Sugars and carbohydrates, such as e.g.glucose, sucrose, lactose, fructose, maltose, molasses, starch andcellulose, oils and fats, such as e.g. soya oil, sunflower oil,groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid,stearic acid and linoleic acid, alcohols, such as e.g. glycerol andethanol, and organic acids, such as e.g. acetic acid, can be used as thesource of carbon. These substances can be used individually or as amixture.

[0150] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0151] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus. The culture medium must furthermorecomprise salts of metals, such as e.g. magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the above-mentioned substances. Precursors of pantothenicacid, such as aspartate, β-alanine, ketoisovalerate, ketopantoic acid orpantoic acid and optionally salts thereof, can moreover be added to theculture medium. The starting substances mentioned can be added to theculture in the form of a single batch, or can be fed in during theculture in a suitable manner.

[0152] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture.

[0153] For the preparation of alkaline earth metal salts of pantothenicacid, in particular the calcium salt or magnesium salt, it is equallypossible to add the suspension or solution of an inorganic compoundcontaining an alkaline earth metal, such as, for example, calciumhydroxide or MgO, or of an organic compound, such as the alkaline earthmetal salt of an organic acid, for example calcium acetate, continuouslyor discontinuously during the fermentation. For this purpose, the cationnecessary for preparation of the desired alkaline earth metal salt ofD-pantothenic acid is introduced into the fermentation broth directly inthe desired amount, preferably in an amount of 0.95 to 1.1 equivalents.

[0154] However, the salts can also be formed after conclusion of thefermentation by addition of the inorganic or organic compounds to thefermentation broth, from which the biomass has optionally been removedbeforehand.

[0155] Antifoams, such as e.g. fatty acid polyglycol esters, can beemployed to control the development of foam. Suitable substances havinga selective action, e.g. antibiotics, can be added to the medium tomaintain the stability of plasmids. To maintain aerobic conditions,oxygen or oxygen-containing gas mixtures, such as e.g. air, areintroduced into the culture. The temperature of the culture is usually15° C. to 95° C., in particular 15° C. to 70° C., preferably 20° C. to55° C., very particularly preferably 30° C. to 50° C. or 30° C. to 45°C. Culturing is continued until a maximum of D-pantothenic acid hasformed. This target is usually reached within 10 hours to 160 hours.

[0156] The D-pantothenic acid or the corresponding salts ofD-pantothenic acid contained in the fermentation broth can then beisolated and purified in accordance with the prior art.

[0157] It is also possible for the fermentation broths comprisingD-pantothenic acid and/or salts thereof preferably first to be freedfrom all or some of the biomass by known separation methods, such as,for example, centrifugation, filtration, decanting or a combinationthereof. However, it is also possible to leave the biomass in itsentirety in the fermentation broth. In general, the suspension orsolution is preferably concentrated and then worked up to a powder, forexample with the aid of a spray dryer or a freeze-drying unit. Thispowder in then in general converted by suitable compacting orgranulating processes, e.g. also build-up granulation, into acoarser-grained, free-flowing, storable and largely dust-free productwith a particle size distribution of preferably 20 to 2000 μm, inparticular 100 to 1400 μm. In the granulation or compacting it isadvantageous to employ conventional organic or inorganic auxiliarysubstances or carriers, such as starch, gelatin, cellulose derivativesor similar substances, such as are conventionally used as binders,gelling agents or thickeners in foodstuffs or feedstuffs processing, orfurther substances, such as, for example, silicas, silicates orstearates.

[0158] Alternatively, the fermentation product, with or without furtherof the conventional fermentation constituents, can be absorbed on to anorganic or inorganic carrier substance which is known and conventionalin feedstuffs processing, such as, for example, silicas, silicates,grits, brans, meals, starches, sugars or others, and/or stabilized withconventional thickeners or binders. Use examples and processes in thiscontext are described in the literature (Die Mühle+Mischfuttertechnik132 (1995) 49, page 817).

[0159] D-Pantothenic acid and/or the desired salt of D-pantothenic acidor a formulation comprising these compounds is optionally added in asuitable process stage during or after the fermentation in order toachieve or establish the content of pantothenic acid desired in theproduct or the desired salt.

[0160] The desired content of pantothenic acid and/or the desired saltis in general in the range from 20 to 80 wt. % (based on the total dryweight).

[0161] The concentration of pantothenic acid can be determined withknown chemical (Velisek; Chromatographic Science 60, 515-560 (1992)) ormicrobiological methods, such as e.g. the Lactobacillus plantarum test(DIFCO MANUAL, 10^(th) Edition, p. 1100-1102; Michigan, USA).

What is claimed is:
 1. Process for the preparation of D-pantothenic acidand/or alkaline earth metal salts thereof or feedstuffs additivescomprising these by fermentation of microorganisms of the Bacillusgroup, in particular those which already produce D-pantothenic acid,wherein a) at least one or more of the nucleotide sequence(s) whichcode(s) for the ybbT-ORF, ywkA-ORF, yjmC-ORF, ytsJ-ORF, mdh gene, cysKgene, iolJ gene, pdhD gene, yuiE-ORF, dhaS gene, adk gene, yusH-ORF,yqhJ-ORF, yqhK-ORF and yqhI-ORF is (are) enhanced, in particularover-expressed, in the microorganisms, b) the D-pantothenic acid and/orsalts thereof are concentrated in the fermentation broth or in the cellsof the microorganisms, and c) after conclusion of the fermentation, thedesired products are isolated, the biomass and/or further constituentsof the fermentation broth being left in the product or optionally beingseparated off completely or in part (0 to 100%).
 2. Process according toclaim 1, wherein the fermentation is carried out in the presence ofalkaline earth metal salts, these being added continuously ordiscontinuously in particular in stoichiometric amounts equivalent tothe D-pantothenic acid formed, and a product comprising alkaline earthmetal salts of D-pantothenic acid or consisting of these being obtained.3. Process according to claim 1 or 2, wherein the microorganismsoriginate from the genus Bacillus, in particular the species Bacillussubtilis.
 4. Process according to claim 1, wherein at the same time oneor more of the genes chosen from the group consisting of the followingis or are additionally enhanced, in particular over-expressed: 4.1 thepanE gene which codes for ketopantoate reductase, 4.2 the open readingframe ylbQ, 4.3 the panB gene which codes for ketopantoate hydroxymethyltransferase, 4.4 the panD gene which codes for aspartate decarboxylase,4.5 the panC gene which codes for pantothenate synthetase, 4.6 the ilvBand ilvN genes which code for acetohydroxy acid synthetase, 4.7 the alsSgene which codes for α-acetolactate synthase, 4.8 the ilvC gene whichcodes for acetohydroxy-acid isomeroreductase, 4.9 the ilvD gene whichcodes for dihydroxy-acid dehydratase, 4.10 the serA gene which codes forphosphoglycerate, dehydrogenase, 4.11 the serC gene which codes forphosphoserine amino transferase, 4.12 the open reading frame ywpJ, and4.13 the glyA gene which codes for serine hydroxymethyl transferase. 5.Process according to claim 1, wherein at the same time one or more ofthe genes chosen from the group consisting of the following is or areadditionally attenuated: 5.1 the protein coded by ywaA-ORF, 5.2 theprotein coded by ybgE-ORF, 5.3 the ansB gene which codes forL-aspartase, 5.4 the alsD gene which codes for acetolactatedecarboxylase, 5.5 the coaA gene which codes for pantothenic acidkinase, and 5.6 the coaX gene which codes for coaX-pantothenic acidkinase.
 6. Process according to claim 1, wherein the over-expression ofone or more of the nucleotide sequence(s) which code(s) for the geneschosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK,iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI is achieved bycarrying out one or more of the measures chosen from the groupconsisting of use of a plasmid vector, optimization of the ribosomebinding site, use of an additional promoter and incorporation of atleast one further gene copy.
 7. Process for the preparation offeedstuffs additives comprising D-pantothenic acid and/or salts thereof,according to claim 1, wherein a) optionally all or some (0 to 100%) ofthe biomass and/or a portion of the constituents is separated off from aD-pantothenic acid-containing fermentation broth obtained byfermentation, b) the mixture obtained in this way is optionallyconcentrated, and c) the feedstuffs additive comprising the pantothenicacid and/or the pantothenate is converted into a free-flowing form bysuitable measures, and d) a free-flowing animal feedstuffs additive witha particle size distribution of 20 to 2000 μm is obtained by suitablemeasures.
 8. Process for the preparation of animal feedstuffs additivesaccording to claim 7 with a content of D-pantothenic acid and/or saltsthereof, chosen from the group consisting of the magnesium or calciumsalt, from fermentation broths, comprising the steps of a) optionallyremoval of water from the fermentation broth (concentration). b) removalof an amount of ≧0 to 100% of the biomass formed during thefermentation, c) optionally addition of one or more of the compoundsmentioned to the fermentation broths obtained according to a) and b),the amount of compounds added being such that the total concentrationthereof in the animal feedstuffs additive is preferably in the rangefrom 20 to 80 wt. %, and d) obtaining of the animal feedstuffs additivein the desired powder or, preferably, granule form.
 9. Process accordingto claim 8, wherein an animal feedstuffs additive with the desiredparticle size is obtained from the fermentation broth, optionally afteraddition of D-pantothenic acid and/or salts thereof and optionally afteraddition of organic and inorganic auxiliaries, by a) drying andcompacting, or b) spray drying, or c) spray drying and granulation, ord) spray drying and build-up granulation.